Protein Folding Handbook, 5 Volume Set

by Buchner, Johannes; Kiefhaber, Thomas

• ISBN13: 9783527307845
• ISBN10: 3527307842
• Edition: 1st
• Format: Hardcover
• Copyright: 2005-03-11
• Publisher: Wiley-VCH
• More Book Details

Summary

This unique handbook contains the expertise from more than 60 research groups, covering the entire range of topics in protein folding - from biophysics to molecular medicine. The first part explains the principles and factors governing protein stability, and how this knowledge may be used to predict folding pathways. It also surveys important techniques used to study the protein folding process, including spectroscopic, chemical and biological techniques. The second part is devoted to protein folding, unfolding, and misfolding in the cellular context, introducing chaperones and other enzymes involved in protein folding, as well as a study of the pathophysiology of misfolded proteins in amyloid and other disease states. The whole is rounded off by a discussion of the possibility of interfering with the protein folding process by genetic engineering. The comprehensiveness and outstanding quality of the carefully selected contents make this the ultimate reference for every scientist with an interest in protein folding.

Author Biography

Johannes Buchner holds the chair of Biotechnology at the Technical University of Munich (Germany). He studied Biology at the University of Regensburg and afterwards joined the group of Ira Pastan at the National Cancer Institute in Bethesda (United States). Returning to Germany, he took a up junior group leader position at Regensburg before becoming full professor at Munich in 1998.<br> Professor Buchner's research is centered on the folding process of proteins and its efficiency in vitro and in vivo. To improve the efficiency of protein production, he has pioneered the biotechnological use of chaperones and holds several patents in this area of research.<br> <br> Thomas Kiefhaber is Professor of Biophysical Chemistry at the Biozentrum, University of Basel (Switzerland). He studied Biology at the University of Regensburg (Germany) and, after completing his PhD in Biophysical Chemistry, spent two years as a post-doc with R. L. Baldwin at Stanford University. Since 1993 he is a member of the faculty of Basel University and was named professor for Biophysical Chemistry in 1999.<br> Professor Kiefhaber's research is centered on the investigation of kinetics and mechanism of protein folding with biophysical methods.

Table of Contents

Preface	lviii
Contributors of Part I	lx
Principles of Protein Stability and Design	1	(376)
Early Days of Studying the Mechanism of Protein Folding	3	(19)
Robert L. Baldwin
Introduction	3	(1)
Two-state Folding	4	(1)
Levinthal's Paradox	5	(1)
The Domain as a Unit of Folding	6	(1)
Detection of Folding Intermediates and Initial Work on the Kinetic Mechanism of Folding	7	(2)
Two Unfolded Forms of RNase A and Explanation by Proline Isomerization	9	(2)
Covalent Intermediates in the Coupled Processes of Disulfide Bond Formation and Folding	11	(1)
Early Stages of Folding Detected by Antibodies and by Hydrogen Exchange	12	(2)
Molten Globule Folding Intermediates	14	(1)
Structures of Peptide Models for Folding Intermediates	15	(7)
Acknowledgments	16	(1)
References	16	(6)
Spectroscopic Techniques to Study Protein Folding and Stability	22	(23)
Franz Schmid
Introduction	22	(1)
Absorbance	23	(6)
Absorbance of Proteins	23	(4)
Practical Considerations for the Measurement of Protein Absorbance	27	(2)
Data Interpretation	29	(1)
Fluorescence	29	(9)
The Fluorescence of Proteins	30	(1)
Energy Transfer and Fluorescence Quenching in a Protein: Barnase	31	(2)
Protein Unfolding Monitored by Fluorescence	33	(3)
Environmental Effects on Tyrosine and Tryptophan Emission	36	(1)
Practical Considerations	37	(1)
Circular Dichroism	38	(7)
CD Spectra of Native and Unfolded Proteins	38	(3)
Measurement of Circular Dichroism	41	(1)
Evaluation of CD Data	42	(1)
References	43	(2)
Denaturation of Proteins by Urea and Guanidine Hydrochloride	45	(25)
C. Nick Pace
Gerald R. Grimsley
J. Martin Scholtz
Historical Perspective	45	(1)
How Urea Denatures Proteins	45	(3)
Linear Extrapolation Method	48	(2)
ΔG(H2O)	50	(5)
m-Values	55	(3)
Concluding Remarks	58	(1)
Experimental Protocols	59	(11)
How to Choose the Best Denaturant for your Study	59	(1)
How to Prepare Denaturant Solutions	59	(1)
How to Determine Solvent Denaturation Curves	60	(2)
Determining a Urea or GdmCl Denaturation Curve	62	(1)
How to Analyze Urea or GdmCl Denaturant Curves	63	(1)
Determining Differences in Stability	64	(1)
Acknowledgments	65	(1)
References	65	(5)
Thermal Unfolding of Proteins Studied by Calorimetry	70	(29)
George I. Makhatadze
Introduction	70	(1)
Two-state Unfolding	71	(5)
Cold Denaturation	76	(1)
Mechanisms of Thermostabilization	77	(2)
Thermodynamic Dissection of Forces Contributing to Protein Stability	79	(5)
Heat Capacity Changes, ΔCp	81	(1)
Enthalpy of Unfolding, ΔH	81	(2)
Entropy of Unfolding, ΔS	83	(1)
Multistate Transitions	84	(8)
Two-state Dimeric Model	85	(1)
Two-state Multimeric Model	86	(1)
Three-state Dimeric Model	86	(2)
Two-state Model with Ligand Binding	88	(2)
Four-state (Two-domain Protein) Model	90	(2)
Experimental Protocols	92	(7)
How to Prepare for DSC Experiments	92	(2)
How to Choose Appropriate Conditions	94	(1)
Critical Factors in Running DSC Experiments	94	(1)
References	95	(4)
Pressure--Temperature Phase Diagrams of Proteins	99	(28)
Wolfgang Doster
Josef Friedrich
Introduction	99	(1)
Basic Aspects of Phase Diagrams of Proteins and Early Experiments	100	(3)
Thermodynamics of Pressure--Temperature Phase Diagrams	103	(7)
Measuring Phase Stability Boundaries with Optical Techniques	110	(6)
Fluorescence Experiments with Cytochrome c	110	(2)
Results	112	(4)
What Do We Learn from the Stability Diagram?	116	(7)
Thermodynamics	116	(1)
Determination of the Equilibrium Constant of Denaturation	117	(3)
Microscopic Aspects	120	(2)
Structural Features of the Pressure-denatured State	122	(1)
Conclusions and Outlook	123	(4)
Acknowledgment	124	(1)
References	124	(3)
Weak Interactions in Protein Folding: Hydrophobic Free Energy, van der Waals Interactions, Peptide Hydrogen Bonds, and Peptide Solvation	127	(36)
Robert L. Baldwin
Introduction	127	(1)
Hydrophobic Free Energy, Burial of Nonpolar Surface and van der Waals Interactions	128	(17)
History	128	(1)
Liquid--Liquid Transfer Model	128	(2)
Relation between Hydrophobic Free Energy and Molecular Surface Area	130	(1)
Quasi-experimental Estimates of the Work of Making a Cavity in Water or in Liquid Alkane	131	(2)
Molecular Dynamics Simulations of the Work of Making Cavities in Water	133	(1)
Dependence of Transfer Free Energy on the Volume of the Solute	134	(2)
Molecular Nature of Hydrophobic Free Energy	136	(1)
Simulation of Hydrophobic Clusters	137	(1)
ΔCp and the Temperature-dependent Thermodynamics of Hydrophobic Free Energy	137	(5)
Modeling Formation of the Hydrophobic Core from Solvation Free Energy and van der Waals Interactions between Nonpolar Residues	142	(2)
Evidence Supporting a Role for van der Waals Interactions in Forming the Hydrophobic Core	144	(1)
Peptide Solvation and the Peptide Hydrogen Bond	145	(18)
History	145	(2)
Solvation Free Energies of Amides	147	(2)
Test of the Hydrogen-Bond Inventory	149	(1)
The Born Equation	150	(1)
Prediction of Solvation Free Energies of Polar Molecules by an Electrostatic Algorithm	150	(1)
Prediction of the Solvation Free Energies of Peptide Groups in Different Backbone Conformations	151	(2)
Predicted Desolvation Penalty for Burial of a Peptide H-bond	153	(1)
Gas--Liquid Transfer Model	154	(2)
Acknowledgments	156	(1)
References	156	(7)
Electrostatics of Proteins: Principles, Models and Applications	163	(38)
Sonja Braun-Sand
Arieh Warshel
Introduction	163	(1)
Historical Perspectives	163	(3)
Electrostatic Models: From Microscopic to Macroscopic Models	166	(7)
All-Atom Models	166	(2)
Dipolar Lattice Models and the PDLD Approach	168	(2)
The PDLD/S-LRA Model	170	(1)
Continuum (Poisson-Boltzmann) and Related Approaches	171	(1)
Effective Dielectric Constant for Charge--Charge Interactions and the GB Model	172	(1)
The Meaning and Use of the Protein Dielectric Constant	173	(3)
Validation Studies	176	(2)
Systems Studied	178	(11)
Solvation Energies of Small Molecules	178	(1)
Calculation of pKa Values of Ionizable Residues	179	(1)
Redox and Electron Transport Processes	180	(1)
Ligand Binding	181	(1)
Enzyme Catalysis	182	(1)
Ion Pairs	183	(1)
Protein--Protein Interactions	184	(1)
Ion Channels	185	(1)
Helix Macrodipoles versus Localized Molecular Dipoles	185	(1)
Folding and Stability	186	(3)
Concluding Remarks	189	(12)
Acknowledgments	190	(1)
References	190	(11)
Protein Conformational Transitions as Seen from the Solvent: Magnetic Relaxation Dispersion Studies of Water, Co-solvent, and Denaturant Interactions with Nonnative Proteins	201	(46)
Bertil Halle
Vladimir P. Denisov
Kristofer Modig
Monika Davidovic
The Role of the Solvent in Protein Folding and Stability	201	(1)
Information Content of Magnetic Relaxation Dispersion	202	(3)
Thermal Perturbations	205	(8)
Heat Denaturation	205	(4)
Cold Denaturation	209	(4)
Electrostatic Perturbations	213	(5)
Solvent Perturbations	218	(15)
Denaturation Induced by Urea	219	(6)
Denaturation Induced by Guanidinium Chloride	225	(3)
Conformational Transitions Induced by Co-solvents	228	(5)
Outlook	233	(1)
Experimental Protocols and Data Analysis	233	(14)
Experimental Methodology	233	(1)
Multiple-field MRD	234	(1)
Field-cycling MRD	234	(1)
Choice of Nuclear Isotope	235	(1)
Data Analysis	236	(1)
Exchange Averaging	236	(1)
Spectral Density Function	237	(2)
Residence Time	239	(1)
19F Relaxation	240	(1)
Coexisting Protein Species	241	(1)
Preferential Solvation	241	(1)
References	242	(5)
Stability and Design of α-Helices	247	(67)
Andrew J. Doig
Neil Errington
Teuku M. Iqbalsyah
Introduction	247	(1)
Structure of the α-Helix	247	(5)
Capping Motifs	248	(2)
Metal Binding	250	(1)
The 310-Helix	251	(1)
The π-Helix	251	(1)
Design of Peptide Helices	252	(9)
Host--Guest Studies	253	(1)
Helix Lengths	253	(1)
The Helix Dipole	253	(1)
Acetylation and Amidation	254	(1)
Side Chain Spacings	255	(1)
Solubility	256	(1)
Concentration Determination	257	(1)
Design of Peptides to Measure Helix Parameters	257	(2)
Helix Templates	259	(1)
Design of 310-Helices	259	(2)
Design of π-helices	261	(1)
Helix Coil Theory	261	(9)
Zimm-Bragg Model	261	(1)
Lifson-Roig Model	262	(3)
The Unfolded State and Polyproline II Helix	265	(1)
Single Sequence Approximation	265	(1)
N- and C-Caps	266	(1)
Capping Boxes	266	(1)
Side-chain Interactions	266	(1)
N1, N2, and N3 Preferences	267	(1)
Helix Dipole	267	(1)
310- and π-Helices	268	(1)
AGADIR	268	(1)
Lomize-Mosberg Model	269	(1)
Extension of the Zimm-Bragg Model	270	(1)
Availability of Helix/Coil Programs	270	(1)
Forces Affecting α-Helix Stability	270	(11)
Helix Interior	270	(3)
Caps	273	(3)
Phosphorylation	276	(1)
Noncovalent Side-chain Interactions	276	(1)
Covalent Side-chain interactions	277	(1)
Capping Motifs	277	(2)
Ionic Strength	279	(1)
Temperature	279	(1)
Trifluoroethanol	279	(1)
pKa Values	280	(1)
Relevance to Proteins	281	(1)
Experimental Protocols and Strategies	281	(33)
Solid Phase Peptide Synthesis (SPPS) Based on the Fmoc Strategy	281	(1)
Equipment and Reagents	281	(2)
Fmoc Deprotection and Coupling	283	(1)
Kaiser Test	284	(1)
Acetylation and Cleavage	285	(1)
Peptide Precipitation	286	(1)
Peptide Purification	286	(1)
Equipment and Reagents	286	(1)
Method	286	(1)
Circular Dichroism	287	(1)
Acquisition of Spectra	288	(1)
Instrumental Considerations	288	(1)
Data Manipulation and Analysis	289	(2)
Protocol for CD Measurement of Helix Content	291	(1)
Aggregation Test for Helical Peptides	291	(1)
Equipment and Reagents	291	(1)
Method	292	(1)
Vibrational Circular Dichroism	292	(1)
NMR Spectroscopy	292	(1)
Nuclear Overhauser Effect	293	(1)
Amide Proton Exchange Rates	294	(1)
13C NMR	294	(1)
Fourier Transform Infrared Spectroscopy	295	(1)
Secondary Structure	295	(1)
Raman Spectroscopy and Raman Optical Activity	296	(2)
pH Titrations	298	(1)
Equipment and Reagents	298	(1)
Method	298	(1)
Acknowledgments	299	(1)
References	299	(15)
Design and Stability of Peptide β-Sheets	314	(29)
Mark S. Searle
Introduction	314	(1)
β-Hairpins Derived from Native Protein Sequences	315	(1)
Role of β-Turns in Nucleating β-Hairpin Folding	316	(3)
Intrinsic φ, ψ Propensities of Amino Acids	319	(2)
Side-chain Interactions and β-Hairpin Stability	321	(9)
Aromatic Clusters Stabilize β-Hairpins	322	(3)
Salt Bridges Enhance Hairpin Stability	325	(5)
Cooperative Interactions in β-Sheet Peptides: Kinetic Barriers to Folding	330	(1)
Quantitative Analysis of Peptide Folding	331	(1)
Thermodynamics of β-Hairpin Folding	332	(2)
Multistranded Antiparallel β-Sheet Peptides	334	(5)
Concluding Remarks: Weak Interactions and Stabilization of Peptide β-Sheets	339	(4)
References	340	(3)
Predicting Free Energy Changes of Mutations in Proteins	343	(34)
Raphael Guerois
Joaquim Mendes
Luis Serrano
Physical Forces that Determine Protein Conformational Stability	343	(10)
Protein Conformational Stability [1]	343	(1)
Structures of the N and D States [2--6]	344	(2)
Studies Aimed at Understanding the Physical Forces that Determine Protein Conformational Stability [1, 2, 8, 19--26]	346	(1)
Forces Determining Conformational Stability [1, 2, 8, 19--27]	346	(1)
Intramolecular Interactions	347	(1)
van der Waals Interactions	347	(1)
Electrostatic Interactions	347	(2)
Conformational Strain	349	(1)
Solvation	350	(1)
Intramolecular Interactions and Solvation Taken Together	350	(1)
Entropy	351	(1)
Cavity Formation	352	(1)
Summary	353	(1)
Methods for the Prediction of the Effect of Point Mutations on in vitro Protein Stability	353	(13)
General Considerations on Protein Plasticity upon Mutation	353	(2)
Predictive Strategies	355	(1)
Methods	356	(1)
From Sequence and Multiple Sequence Alignment Analysis	356	(1)
Statistical Analysis of the Structure Databases	356	(1)
Helix/Coil Transition Model	357	(2)
Physicochemical Method Based on Protein Engineering Experiments	359	(5)
Methods Based only on the Basic Principles of Physics and Thermodynamics	364	(2)
Mutation Effects on in vivo Stability	366	(2)
The N-terminal Rule	366	(1)
The C-terminal Rule	367	(1)
PEST Signals	368	(1)
Mutation Effects on Aggregation	368	(9)
References	369	(8)
Dynamics and Mechanisms of Protein Folding Reactions	377	(957)
Kinetic Mechanisms in Protein Folding	379	(32)
Annett Bachmann
Thomas Kiefhaber
Introduction	379	(1)
Analysis of Protein Folding Reactions using Simple Kinetic Models	379	(1)
General Treatment of Kinetic Data	380	(1)
Two-state Protein Folding	380	(4)
Complex Folding Kinetics	384	(1)
Heterogeneity in the Unfolded State	384	(4)
Folding through Intermediates	388	(3)
Rapid Pre-equilibria	391	(2)
Folding through an On-pathway High-energy Intermediate	393	(1)
A Case Study: the Mechanism of Lysozyme Folding	394	(1)
Lysozyme Folding at pH 5.2 and Low Salt Concentrations	394	(4)
Lysozyme Folding at pH 9.2 or at High Salt Concentrations	398	(3)
Non-exponential Kinetics	401	(1)
Conclusions and Outlook	401	(1)
Protocols -- Analytical Solutions of Three-state Protein Folding Models	402	(1)
Triangular Mechanism	402	(1)
On-pathway Intermediate	403	(1)
Off-pathway Mechanism	404	(1)
Folding Through an On-pathway High-Energy Intermediate	404	(2)
Acknowledgments	406	(1)
References	406	(5)
Characterization of Protein Folding Barriers with Rate Equilibrium Free Energy Relationships	411	(34)
Thomas Kiefhaber
Ignacio E. Sanchez
Annett Bachmann
Introduction	411	(1)
Rate Equilibrium Free Energy Relationships	411	(3)
Linear Rate Equilibrium Free Energy Relationships in Protein Folding	414	(4)
Properties of Protein Folding Transition States Derived from Linear REFERs	418	(2)
Nonlinear Rate Equilibrium Free Energy Relationships in Protein Folding	420	(1)
Self-Interaction and Cross-Interaction Parameters	420	(4)
Hammond and Anti-Hammond Behavior	424	(1)
Sequential and Parallel Transition States	425	(3)
Ground State Effects	428	(4)
Experimental Results on the Shape of Free Energy Barriers in Protein Folding	432	(1)
Broadness of Free Energy Barriers	432	(5)
Parallel Pathways	437	(1)
Folding in the Absence of Enthalpy Barriers	438	(1)
Conclusions and Outlook	438	(1)
Acknowledgments	439	(1)
References	439	(6)
A Guide to Measuring and Interpreting φ-values	445	(9)
Nicholas R. Guydosh
Alan R. Fersht
Introduction	445	(1)
Basic Concept of φ-Value Analysis	445	(3)
Further Interpretation of φ	448	(2)
Techniques	450	(2)
Conclusions	452	(2)
References	452	(2)
Fast Relaxation Methods	454	(37)
Martin Gruebele
Introduction	454	(1)
Techniques	455	(11)
Fast Pressure-Jump Experiments	455	(1)
Fast Resistive Heating Experiments	456	(1)
Fast Laser-induced Relaxation Experiments	457	(1)
Laser Photolysis	457	(1)
Electrochemical Jumps	458	(1)
Laser-induced pH Jumps	458	(1)
Covalent Bond Dissociation	459	(1)
Chromophore Excitation	460	(1)
Laser Temperature Jumps	460	(1)
Multichannel Detection Techniques for Relaxation Studies	461	(1)
Small Angle X-ray Scattering or Light Scattering	462	(1)
Direct Absorption Techniques	463	(1)
Circular Dichroism and Optical Rotatory Dispersion	464	(1)
Raman and Resonance Raman Scattering	464	(1)
Intrinsic Fluorescence	465	(1)
Extrinsic Fluorescence	465	(1)
Protein Folding by Relaxation	466	(8)
Transition State Theory, Energy Landscapes, and Fast Folding	466	(4)
Viscosity Dependence of Folding Motions	470	(1)
Resolving Burst Phases	471	(1)
Fast Folding and Unfolded Proteins	472	(1)
Experiment and Simulation	472	(2)
Summary	474	(1)
Experimental Protocols	475	(16)
Design Criteria for Laser Temperature Jumps	475	(1)
Design Criteria for Fast Single-Shot Detection Systems	476	(1)
Designing Proteins for Fast Relaxation Experiments	477	(1)
Linear Kinetic, Nonlinear Kinetic, and Generalized Kinetic Analysis of Fast Relaxation	477	(1)
The Reaction D F in the Presence of a Barrier	477	(1)
The Reaction 2A A2 in the Presence of a Barrier	478	(1)
The Reaction D F at Short Times or over Low Barriers	479	(1)
Relaxation Data Analysis by Linear Decomposition	480	(1)
Singular Value Decomposition (SVD)	480	(1)
Χ-Analysis	481	(1)
Acknowledgments	481	(1)
References	482	(9)
Early Events in Protein Folding Explored by Rapid Mixing Methods	491	(45)
Heinrich Roder
Kosuke Maki
Ramil F. Latypov
Hong Cheng
M. C. Ramachandra Shastry
Importance of Kinetics for Understanding Protein Folding	491	(1)
Burst-phase Signals in Stopped-flow Experiments	492	(2)
Turbulent Mixing	494	(1)
Detection Methods	495	(7)
Tryptophan Fluorescence	495	(3)
ANS Fluorescence	498	(1)
FRET	499	(2)
Continuous-flow Absorbance	501	(1)
Other Detection Methods used in Ultrafast Folding Studies	502	(1)
A Quenched-Flow Method for H-D Exchange Labeling Studies on the Microsecond Time Scale	502	(3)
Evidence for Accumulation of Early Folding Intermediates in Small Proteins	505	(10)
B1 Domain of Protein G	505	(3)
Ubiquitin	508	(4)
Cytochrome c	512	(3)
Significance of Early Folding Events	515	(21)
Barrier-limited Folding vs. Chain Diffusion	515	(1)
Chain Compaction: Random Collapse vs. Specific Folding	516	(1)
Kinetic Role of Early Folding Intermediates	517	(3)
Broader Implications	520	(1)
Appendix	521	(1)
Design and Calibration of Rapid Mixing Instruments	521	(1)
Stopped-flow Equipment	521	(3)
Continuous-flow Instrumentation	524	(4)
Acknowledgments	528	(1)
References	528	(8)
Kinetic Protein Folding Studies using NMR Spectroscopy	536	(37)
Markus Zeeb
Jochen Balbach
Introduction	536	(2)
Following Slow Protein Folding Reactions in Real Time	538	(7)
Two-dimensional Real-time NMR Spectroscopy	545	(5)
Dynamic and Spin Relaxation NMR for Quantifying Microsecond-to-Millisecond Folding Rates	550	(5)
Conclusions and Future Directions	555	(1)
Experimental Protocols	556	(17)
How to Record and Analyze 1D Real-time NMR Spectra	556	(1)
Acquisition	556	(1)
Processing	557	(1)
Analysis	557	(1)
Analysis of 1D Real-time Diffusion Experiments	558	(1)
How to Extract Folding Rates from 1D Spectra by Line Shape Analysis	559	(1)
Acquisition	560	(1)
Processing	560	(1)
Analysis	561	(1)
How to Extract Folding Rates from 2D Real-time NMR Spectra	562	(1)
Acquisition	563	(1)
Processing	563	(1)
Analysis	563	(2)
How to Analyze Heteronuclear NMR Relaxation and Exchange Data	565	(1)
Acquisition	566	(1)
Processing	567	(1)
Analysis	567	(2)
Acknowledgments	569	(1)
References	569	(4)
Fluorescence Resonance Energy Transfer (FRET) and Single Molecule Fluorescence Detection Studies of the Mechanism of Protein Folding and Unfolding	573	(61)
Elisha Haas
Abbreviations	573	(1)
Introduction	573	(1)
What are the Main Aspects of the Protein Folding Problem that can be Addressed by Methods Based on FRET Measurements?	574	(2)
The Three Protein Folding Problems	574	(1)
The Chain Entropy Problem	574	(1)
The Function Problem: Conformational Fluctuations	575	(1)
Theoretical Background	576	(10)
Nonradiative Excitation Energy Transfer	576	(1)
What is FRET? The Singlet--Singlet Excitation Transfer	577	(1)
Rate of Nonradiative Excitation Energy Transfer within a Donor--Acceptor Pair	578	(5)
The Orientation Factor	583	(1)
How to Determine and Control the Value of R?	584	(1)
Index of Refraction n	584	(2)
The Donor Quantum Yield ΦD	586	(1)
The Spectral Overlap Integral J	586	(1)
Determination of Intramolecular Distances in Protein Molecules using FRET Measurements	586	(8)
Single Distance between Donor and Acceptor	587	(1)
Method 1: Steady State Determination of Decrease of Donor Emission	587	(1)
Method 2: Acceptor Excitation Spectroscopy	588	(1)
Time-resolved Methods	588	(1)
Determination of E from Donor Fluorescence Decay Rates	589	(1)
Determination of Acceptor Fluorescence Lifetime	589	(1)
Determination of Intramolecular Distance Distributions	590	(2)
Evaluation of the Effect of Fast Conformational Fluctuations and Determination of Intramolecular Diffusion Coefficients	592	(2)
Experimental Challenges in the Implementation of FRET Folding Experiments	594	(6)
Optimized Design and Preparation of Labeled Protein Samples for FRET Folding Experiments	594	(1)
Strategies for Site-specific Double Labeling of Proteins	595	(1)
Preparation of Double-labeled Mutants Using Engineered Cysteine Residues (strategy 4)	596	(3)
Possible Pitfalls Associated with the Preparation of Labeled Protein Samples for FRET Folding Experiments	599	(1)
Experimental Aspects of Folding Studies by Distance Determination Based on FRET Measurements	600	(3)
Steady State Determination of Transfer Efficiency	600	(1)
Donor Emission	600	(1)
Acceptor Excitation Spectroscopy	601	(1)
Time-resolved Measurements	601	(2)
Data Analysis	603	(4)
Rigorous Error Analysis	606	(1)
Elimination of Systematic Errors	606	(1)
Applications of trFRET for Characterization of Unfolded and Partially Folded Conformations of Globular Proteins under Equilibrium Conditions	607	(4)
Bovine Pancreatic Trypsin Inhibitor	607	(1)
The Loop Hypothesis	608	(1)
RNase A	609	(2)
Staphylococcal Nuclease	611	(1)
Unfolding Transition via Continuum of Native-like Forms	611	(1)
The Third Folding Problem: Domain Motions and Conformational Fluctuations of Enzyme Molecules	611	(2)
Single Molecule FRET-detected Folding Experiments	613	(2)
Principles of Applications of Single Molecule FRET Spectroscopy in Folding Studies	615	(4)
Design and Analysis of Single Molecule FRET Experiments	615	(1)
How is Single Molecule FRET Efficiency Determined?	615	(2)
The Challenge of Extending the Length of the Time Trajectories	617	(1)
Distance and Time Resolution of the Single Molecule FRET Folding Experiments	618	(1)
Folding Kinetics	619	(6)
Steady State and trFRET-detected Folding Kinetics Experiments	619	(1)
Steady State Detection	619	(2)
Time-resolved FRET Detection of Rapid Folding Kinetics: the ``Double Kinetics'' Experiment	621	(1)
Multiple Probes Analysis of the Folding Transition	622	(3)
Concluding Remarks	625	(9)
Acknowledgments	626	(1)
References	627	(7)
Application of Hydrogen Exchange Kinetics to Studies of Protein Folding	634	(39)
Kaare Teilum
Birthe B. Kragelund
Flemming M. Poulsen
Introduction	634	(4)
The Hydrogen Exchange Reaction	638	(3)
Calculating the Intrinsic Hydrogen Exchange Rate Constant, k/int	638	(3)
Protein Dynamics by Hydrogen Exchange in Native and Denaturing Conditions	641	(10)
Mechanisms of Exchange	642	(1)
Local Opening and Closing Rates from Hydrogen Exchange Kinetics	642	(1)
The General Amide Exchange Rate Expression -- the Linderstrøm-Lang Equation	643	(1)
Limits to the General Rate Expression -- EX1 and EX2	644	(2)
The Range between the EX1 and EX2 Limits	646	(1)
Identification of Exchange Limit	646	(1)
Global Opening and Closing Rates and Protein Folding	647	(1)
The ``Native State Hydrogen Exchange'' Strategy	648	(2)
Localization of Partially Unfolded States, PUFs	650	(1)
Hydrogen Exchange as a Structural Probe in Kinetic Folding Experiments	651	(10)
Protein Folding/Hydrogen Exchange Competition	652	(4)
Hydrogen Exchange Pulse Labeling	656	(1)
Protection Factors in Folding Intermediates	657	(2)
Kinetic Intermediate Structures Characterized by Hydrogen Exchange	659	(2)
Experimental Protocols	661	(12)
How to Determine Hydrogen Exchange Kinetics at Equilibrium	661	(1)
Equilibrium Hydrogen Exchange Experiments	661	(1)
Determination of Segmental Opening and Closing Rates, k/op and k/cl	662	(1)
Determination of ΔGfluc, m, and ΔGunf	662	(1)
Planning a Hydrogen Exchange Folding Experiment	662	(1)
Determine a Combination of tpulse and pHpulse	662	(1)
Setup Quench Flow Apparatus	662	(1)
Prepare Deuterated Protein and Chemicals	663	(1)
Prepare Buffers and Unfolded Protein	663	(1)
Check pH in the Mixing Steps	664	(1)
Sample Mixing and Preparation	664	(1)
Data Analysis	664	(1)
Acknowledgments	665	(1)
References	665	(8)
Studying Protein Folding and Aggregation by Laser Light Scattering	673	(37)
Klaus Gast
Andreas J. Modler
Introduction	673	(1)
Basic Principles of Laser Light Scattering	674	(6)
Light Scattering by Macromolecular Solutions	674	(2)
Molecular Parameters Obtained from Static Light Scattering (SLS)	676	(2)
Molecular Parameters Obtained from Dynamic Light Scattering (DLS)	678	(2)
Advantages of Combined SLS and DLS Experiments	680	(1)
Laser Light Scattering of Proteins in Different Conformational States -- Equilibrium Folding/Unfolding Transitions	680	(9)
General Considerations, Hydrodynamic Dimensions in the Natively Folded State	680	(2)
Changes in the Hydrodynamic Dimensions during Heat-induced Unfolding	682	(1)
Changes in the Hydrodynamic Dimensions upon Cold Denaturation	683	(1)
Denaturant-induced Changes of the Hydrodynamic Dimensions	684	(1)
Acid-induced Changes of the Hydrodynamic Dimensions	685	(1)
Dimensions in Partially Folded States -- Molten Globules and Fluoroalcohol-induced States	686	(1)
Comparison of the Dimensions of Proteins in Different Conformational States	687	(1)
Scaling Laws for the Native and Highly Unfolded States, Hydrodynamic Modeling	687	(2)
Studying Folding Kinetics by Laser Light Scattering	689	(3)
General Considerations, Attainable Time Regions	689	(1)
Hydrodynamic Dimensions of the Kinetic Molten Globule of Bovine α-Lactalbumin	690	(1)
RNase A is Only Weakly Collapsed During the Burst Phase of Folding	691	(1)
Misfolding and Aggregation Studied by Laser Light Scattering	692	(6)
Overview: Some Typical Light Scattering Studies of Protein Aggregation	692	(1)
Studying Misfolding and Amyloid Formation by Laser Light Scattering	693	(1)
Overview: Initial States, Critical Oligomers, Protofibrils, Fibrils	693	(1)
Aggregation Kinetics of Aβ Peptides	694	(1)
Kinetics of Oligomer and Fibril Formation of PGK and Recombinant Hamster Prion Protein	695	(3)
Mechanisms of Misfolding and Misassembly, Some General Remarks	698	(1)
Experimental Protocols	698	(12)
Laser Light Scattering Instrumentation	698	(1)
Basic Experimental Set-up, General Requirements	698	(2)
Supplementary Measurements and Useful Options	700	(1)
Commercially Available Light Scattering Instrumentation	701	(1)
Experimental Protocols for the Determination of Molecular Mass and Stokes Radius of a Protein in a Particular Conformational State	701	(1)
Protocol 1	702	(2)
Protocol 2	704	(1)
Acknowledgments	704	(1)
References	704	(6)
Conformational Properties of Unfolded Proteins	710	(27)
Patrick J. Fleming
George D. Rose
Introduction	710	(1)
Unfolded vs. Denatured Proteins	710	(1)
Early History	711	(1)
The Random Coil	712	(9)
The Random Coil -- Theory	713	(3)
The Random Coil Model Prompts Three Questions	716	(1)
The Folding Funnel	716	(1)
Transition State Theory	717	(1)
Other Examples	717	(1)
Implicit Assumptions from the Random Coil Model	718	(1)
The Random Coil -- Experiment	718	(1)
Intrinsic Viscosity	719	(1)
SAXS and SANS	720	(1)
Questions about the Random Coil Model	721	(9)
Questions from Theory	722	(1)
The Flory Isolated-pair Hypothesis	722	(2)
Structure vs. Energy Duality	724	(1)
The ``Rediscovery'' of Polyproline II Conformation	724	(2)
PII in Unfolded Peptides and Proteins	726	(1)
Questions from Experiment	727	(1)
Residual Structure in Denatured Proteins and Peptides	727	(1)
The Reconciliation Problem	728	(1)
Organization in the Unfolded State -- the Entropic Conjecture	728	(1)
Steric Restrictions beyond the Dipeptide	729	(1)
Future Directions	730	(7)
Acknowledgments	731	(1)
References	731	(6)
Conformation and Dynamics of Nonnative States of Proteins studied by NMR Spectroscopy	737	(72)
Julia Wirmer
Christian Schlorb
Harald Schwalbe
Introduction	737	(3)
Structural Diversity of Polypeptide Chains	737	(2)
Intrinsically Unstructured and Natively Unfolded Proteins	739	(1)
Prerequisites: NMR Resonance Assignment	740	(4)
NMR Parameters	744	(24)
Chemical shifts δ	745	(1)
Conformational Dependence of Chemical Shifts	745	(1)
Interpretation of Chemical Shifts in the Presence of Conformational Averaging	746	(2)
J Coupling Constants	748	(1)
Conformational Dependence of J Coupling Constants	748	(2)
Interpretation of J Coupling Constants in the Presence of Conformational Averaging	750	(1)
Relaxation: Homonuclear NOEs	750	(1)
Distance Dependence of Homonuclear NOEs	750	(4)
Interpretation of Homonuclear NOEs in the Presence of Conformational Averaging	754	(3)
Heteronuclear Relaxation (15N R1, R2, hetNOE)	757	(1)
Correlation Time Dependence of Heteronuclear Relaxation Parameters	757	(2)
Dependence on Internal Motions of Heteronuclear Relaxation Parameters	759	(1)
Residual Dipolar Couplings	760	(1)
Conformational Dependence of Residual Dipolar Couplings	760	(3)
Interpretation of Residual Dipolar Couplings in the Presence of Conformational Averaging	763	(2)
Diffusion	765	(1)
Paramagnetic Spin Labels	766	(1)
H/D Exchange	767	(1)
Photo-CIDNP	767	(1)
Model for the Random Coil State of a Protein	768	(3)
Nonnative States of Proteins: Examples from Lysozyme, α-Lactalbumin, and Ubiquitin	771	(22)
Backbone Conformation	772	(1)
Interpretation of Chemical Shifts	772	(2)
Interpretation of NOEs	774	(6)
Interpretation of J Coupling Constants	780	(4)
Side-chain Conformation	784	(1)
Interpretation of J Coupling Constants	784	(2)
Backbone Dynamics	786	(1)
Interpretation of 15N Relaxation Rates	786	(7)
Summary and Outlook	793	(16)
Acknowledgments	794	(1)
References	794	(15)
Dynamics of Unfolded Polypeptide Chains	809	(47)
Beat Fierz
Thomas Kiefhaber
Introduction	809	(1)
Equilibrium Properties of Chain Molecules	809	(4)
The Freely Jointed Chain	810	(1)
Chain Stiffness	810	(1)
Polypeptide Chains	811	(1)
Excluded Volume Effects	812	(1)
Theory of Polymer Dynamics	813	(3)
The Langevin Equation	813	(1)
Rouse Model and Zimm Model	814	(1)
Dynamics of Loop Closure and the Szabo-Schulten-Schulten Theory	815	(1)
Experimental Studies on the Dynamics in Unfolded Polypeptide Chains	816	(21)
Experimental Systems for the Study of Intrachain Diffusion	816	(1)
Early Experimental Studies	816	(5)
Triplet Transfer and Triplet Quenching Studies	821	(4)
Fluorescence Quenching	825	(1)
Experimental Results on Dynamic Properties of Unfolded Polypeptide Chains	825	(1)
Kinetics of Intrachain Diffusion	826	(1)
Effect of Loop Size on the Dynamics in Flexible Polypeptide Chains	826	(3)
Effect of Amino Acid Sequence on Chain Dynamics	829	(2)
Effect of the Solvent on Intrachain Diffusion	831	(2)
Effect of Solvent Viscosity on Intrachain Diffusion	833	(1)
End-to-end Diffusion vs. Intrachain Diffusion	834	(1)
Chain Diffusion in Natural Protein Sequences	834	(3)
Implications for Protein Folding Kinetics	837	(7)
Rate of Contact Formation during the Earliest Steps in Protein Folding	837	(2)
The Speed Limit of Protein Folding vs. the Pre-exponential Factor	839	(1)
Contributions of Chain Dynamics to Rate- and Equilibrium Constants for Protein Folding Reactions	840	(4)
Conclusions and Outlook	844	(1)
Experimental Protocols and Instrumentation	844	(12)
Properties of the Electron Transfer Probes and Treatment of the Transfer Kinetics	845	(2)
Test for Diffusion-controlled Reactions	847	(1)
Determination of Bimolecular Quenching or Transfer Rate Constants	847	(1)
Testing the Viscosity Dependence	848	(1)
Determination of Activation Energy	848	(1)
Instrumentation	849	(1)
Acknowledgments	849	(1)
References	849	(7)
Equilibrium and Kinetically Observed Molten Globule States	856	(28)
Kosuke Maki
Kiyoto Kamagata
Kunihiro Kuwajima
Introduction	856	(2)
Equilibrium Molten Globule State	858	(4)
Structural Characteristics of the Molten Globule State	858	(1)
Typical Examples of the Equilibrium Molten Globule State	859	(1)
Thermodynamic Properties of the Molten Globule State	860	(2)
The Kinetically Observed Molten Globule State	862	(4)
Observation and Identification of the Molten Globule State in Kinetic Refolding	862	(1)
Kinetics of Formation of the Early Folding Intermediates	863	(1)
Late Folding Intermediates and Structural Diversity	864	(1)
Evidence for the On-pathway Folding Intermediate	865	(1)
Two-stage Hierarchical Folding Funnel	866	(1)
Unification of the Folding Mechanism between Non-two-state and Two-state Proteins	867	(5)
Statistical Analysis of the Folding Data of Non-two-state and Two-state Proteins	868	(2)
A Unified Mechanism of Protein Folding: Hierarchy	870	(1)
Hidden Folding Intermediates in Two-state Proteins	871	(1)
Practical Aspects of the Experimental Study of Molten Globules	872	(12)
Observation of the Equilibrium Molten Globule State	872	(1)
Two-state Unfolding Transition	872	(2)
Multi-state (Three-state) Unfolding Transition	874	(2)
Burst-phase Intermediate Accumulated during the Dead Time of Refolding Kinetics	876	(1)
Testing the Identity of the Molten Globule State with the Burst-Phase Intermediate	877	(2)
References	879	(5)
Alcohol- and Salt-induced Partially Folded Intermediates	884	(32)
Daizo Hamada
Yuji Goto
Introduction	884	(2)
Alcohol-induced Intermediates of Proteins and Peptides	886	(7)
Formation of Secondary Structures by Alcohols	888	(1)
Alcohol-induced Denaturation of Proteins	888	(1)
Formation of Compact Molten Globule States	889	(1)
Example: β-Lactoglobulin	890	(3)
Mechanism of Alcohol-induced Conformational Change	893	(3)
Effects of Alcohols on Folding Kinetics	896	(3)
Salt-induced Formation of the Intermediate States	899	(5)
Acid-denatured Proteins	899	(1)
Acid-induced Unfolding and Refolding Transitions	900	(4)
Mechanism of Salt-induced Conformational Change	904	(2)
Generality of the Salt Effects	906	(10)
Conclusion	907	(1)
References	908	(8)
Prolyl Isomerization in Protein Folding	916	(30)
Franz Schmid
Introduction	916	(1)
Prolyl Peptide Bonds	917	(1)
Prolyl Isomerizations as Rate-determining Steps of Protein Folding	918	(6)
The Discovery of Fast and Slow Refolding Species	918	(1)
Detection of Proline-limited Folding Processes	919	(2)
Proline-limited Folding Reactions	921	(2)
Interrelation between Prolyl Isomerization and Conformational Folding	923	(1)
Examples of Proline-limited Folding Reactions	924	(5)
Ribonuclease A	924	(2)
Ribonuclease T1	926	(2)
The Structure of a Folding Intermediate with an Incorrect Prolyl Isomer	928	(1)
Native-state Prolyl Isomerizations	929	(1)
Nonprolyl Isomerizations in Protein Folding	930	(2)
Catalysis of Protein Folding by Prolyl Isomerases	932	(4)
Prolyl Isomerases as Tools for Identifying Proline-limited Folding Steps	932	(1)
Specificity of Prolyl Isomerases	933	(1)
The Trigger Factor	934	(1)
Catalysis of Prolyl Isomerization During de novo Protein Folding	935	(1)
Concluding Remarks	936	(1)
Experimental Protocols	936	(10)
Slow Refolding Assays (``Double Jumps'') to Measure Prolyl Isomerizations in an Unfolded Protein	936	(1)
Guidelines for the Design of Double Jump Experiments	937	(1)
Formation of U/s Species after Unfolding of RNase A	938	(1)
Slow Unfolding Assays for Detecting and Measuring Prolyl Isomerizations in Refolding	938	(1)
Practical Considerations	939	(1)
Kinetics-of the Formation of Fully Folded IIHY-G3P* Molecules	939	(1)
References	939	(7)
Folding and Disulfide Formation	946	(19)
Margherita Ruoppolo
Piero Pucci
Gennaro Marino
Chemistry of the Disulfide Bond	946	(1)
Trapping Protein Disulfides	947	(1)
Mass Spectrometric Analysis of Folding Intermediates	948	(1)
Mechanism(s) of Oxidative Folding so Far -- Early and Late Folding Steps	949	(1)
Emerging Concepts from Mass Spectrometric Studies	950	(6)
Three-fingered Toxins	951	(2)
RNase A	953	(2)
Antibody Fragments	955	(1)
Human Nerve Growth Factor	956	(1)
Unanswered Questions	956	(1)
Concluding Remarks	957	(1)
Experimental Protocols	957	(8)
How to Prepare Folding Solutions	957	(1)
How to Carry Out Folding Reactions	958	(1)
How to Choose the Best Mass Spectrometric Equipment for Your Study	959	(1)
How to Perform Electrospray (ES)MS Analysis	959	(1)
How to Perform Matrix-assisted Laser Desorption Ionization (MALDI) MS Analysis	960	(1)
References	961	(4)
Concurrent Association and Folding of Small Oligomeric Proteins	965	(33)
Hans Rudolf Bosshard
Introduction	965	(1)
Experimental Methods Used to Follow the Folding of Oligomeric Proteins	966	(3)
Equilibrium Methods	966	(2)
Kinetic Methods	968	(1)
Dimeric Proteins	969	(14)
Two-state Folding of Dimeric Proteins	970	(1)
Examples of Dimeric Proteins Obeying Two-state Folding	971	(7)
Folding of Dimeric Proteins through Intermediate States	978	(5)
Trimeric and Tetrameric Proteins	983	(3)
Concluding Remarks	986	(12)
Appendix -- Concurrent Association and Folding of Small Oligomeric Proteins	987	(1)
Equilibrium Constants for Two-state Folding	988	(1)
Homooligomeric Protein	988	(1)
Heterooligomeric Protein	989	(1)
Calculation of Thermodynamic Parameters from Equilibrium Constants	990	(1)
Basic Thermodynamic Relationships	990	(1)
Linear Extrapolation of Denaturant Unfolding Curves of Two-state Reaction	990	(1)
Calculation of the van't Hoff Enthalpy Change from Thermal Unfolding Data	990	(1)
Calculation of the van't Hoff Enthalpy Change from the Concentration-dependence of Tm	991	(1)
Extrapolation of Thermodynamic Parameters to Different Temperatures: Gibbs-Helmholtz Equation	991	(1)
Kinetics of Reversible Two-state Folding and Unfolding: Integrated Rate Equations	992	(1)
Two-state Folding of Dimeric Protein	992	(1)
Two-state Unfolding of Dimeric Protein	992	(1)
Reversible Two-state Folding and Unfolding	993	(1)
Homodimeric protein	993	(1)
Heterodimeric protein	993	(1)
Kinetics of Reversible Two-state Folding: Relaxation after Disturbance of a Pre-existing Equilibrium (Method of Bernasconi)	994	(1)
Acknowledgments	995	(1)
References	995	(3)
Folding of Membrane Proteins	998	(34)
Lukas K. Tamm
Heedeok Hong
Introduction	998	(2)
Thermodyamics of Residue Partitioning into Lipid Bilayers	1000	(1)
Stability of β-Barrel Proteins	1001	(8)
Stability of Helical Membrane Proteins	1009	(1)
Helix and Other Lateral Interactions in Membrane Proteins	1010	(2)
The Membrane Interface as an Important Contributor to Membrane Protein Folding	1012	(1)
Membrane Toxins as Models for Helical Membrane Protein Insertion	1013	(2)
Mechanisms of β-Barrel Membrane Protein Folding	1015	(1)
Experimental Protocols	1016	(16)
SDS Gel Shift Assay for Heat-modifiable Membrane Proteins	1016	(1)
Reversible Folding and Unfolding Protocol Using OmpA as an Example	1016	(2)
Tryptophan Fluorescence and Time-resolved Distance Determination by Tryptophan Fluorescence Quenching	1018	(1)
TDFQ Protocol for Monitoring the Translocation of Tryptophans across Membranes	1019	(1)
Circular Dichroism Spectroscopy	1020	(2)
Fourier Transform Infrared Spectroscopy	1022	(1)
Protocol for Obtaining Conformation and Orientation of Membrane Proteins and Peptides by Polarized ATR-FTIR Spectroscopy	1023	(2)
Acknowledgments	1025	(1)
References	1025	(7)
Protein Folding Catalysis by Pro-domains	1032	(27)
Philip N. Bryan
Introduction	1032	(1)
Bimolecular Folding Mechanisms	1033	(1)
Structures of Reactants and Products	1033	(6)
Structure of Free SBT	1033	(3)
Structure of SBT/Pro-domain Complex	1036	(1)
Structure of Free ALP	1037	(1)
Structure of the ALP/Pro-domain Complex	1037	(2)
Stability of the Mature Protease	1039	(3)
Stability of ALP	1039	(1)
Stability of Subtilisin	1040	(2)
Analysis of Pro-domain Binding to the Folded Protease	1042	(1)
Analysis of Folding Steps	1043	(3)
Why are Pro-domains Required for Folding?	1046	(1)
What is the Origin of High Cooperativity?	1047	(1)
How Does the Pro-domain Accelerate Folding?	1048	(1)
Are High Kinetic Stability and Facile Folding Mutually Exclusive?	1049	(1)
Experimental Protocols for Studying SBT Folding	1049	(10)
Fermentation and Purification of Active Subtilisin	1049	(1)
Fermentation and Purification of Facile-folding Ala221 Subtilisin from E. coli	1050	(1)
Mutagenesis and Protein Expression of Pro-domain Mutants	1051	(1)
Purification of Pro-domain	1052	(1)
Kinetics of Pro-domain Binding to Native SBT	1052	(1)
Kinetic Analysis of Pro-domain Facilitated Subtilisin Folding	1052	(1)
Single Mixing	1052	(1)
Double Jump: Renaturation--Denaturation	1053	(1)
Double Jump: Denaturation--Renaturation	1053	(1)
Triple Jump: Denaturation--Renaturation--Denaturation	1054	(1)
References	1054	(5)
The Thermodynamics and Kinetics of Collagen Folding	1059	(52)
Hans Peter Bachinger
Jurgen Engel
Introduction	1059	(3)
The Collagen Family	1059	(1)
Biosynthesis of Collagens	1060	(1)
The Triple Helical Domain in Collagens and Other Proteins	1061	(1)
N- and C-Propeptide, Telopeptides, Flanking Coiled-Coil Domains	1061	(1)
Why is the Folding of the Triple Helix of Interest?	1061	(1)
Thermodynamics of Collagen Folding	1062	(19)
Stability of the Triple Helix	1062	(1)
The Role of Posttranslational Modifications	1063	(1)
Energies Involved in the Stability of the Triple Helix	1063	(3)
Model Peptides Forming the Collagen Triple Helix	1066	(1)
Type of Peptides	1066	(1)
The All-or-none Transition of Short Model Peptides	1066	(3)
Thermodynamic Parameters for Different Model Systems	1069	(6)
Contribution of Different Tripeptide Units to Stability	1075	(1)
Crystal and NMR Structures of Triple Helices	1076	(1)
Conformation of the Randomly Coiled Chains	1077	(1)
Model Studies with Isomers of Hydroxyproline and Fluoroproline	1078	(1)
Cis trans Equilibria of Peptide Bonds	1079	(1)
Interpretations of Stabilities on a Molecular Level	1080	(1)
Kinetics of Triple Helix Formation	1081	(30)
Properties of Collagen Triple Helices that Influence Kinetics	1081	(1)
Folding of Triple Helices from Single Chains	1082	(1)
Early Work	1082	(1)
Concentration Dependence of the Folding of (PPG)10 and (POG)10	1082	(3)
Model Mechanism of the Folding Kinetics	1085	(2)
Rate Constants of Nucleation and Propagation	1087	(1)
Host--guest Peptides and an Alternative Kinetics Model	1088	(1)
Triple Helix Formation from Linked Chains	1089	(1)
The Short N-terminal Triple Helix of Collagen III in Fragment Col1--3	1089	(1)
Folding of the Central Long Triple Helix of Collagen III	1090	(2)
The Zipper Model	1092	(5)
Designed Collagen Models with Chains Connected by a Disulfide Knot or by Trimerizing Domains	1097	(1)
Disulfide-linked Model Peptides	1097	(1)
Model Peptides Linked by a Foldon Domain	1098	(1)
Collagen Triple Helix Formation can be Nucleated at either End	1098	(1)
Hysteresis of Triple Helix Formation	1099	(1)
Influence of cis--trans Isomerase and Chaperones	1100	(1)
Mutations in Collagen Triple Helices Affect Proper Folding	1101	(1)
References	1101	(10)
Unfolding Induced by Mechanical Force	1111	(32)
Jane Clarke
Phil M. Williams
Introduction	1111	(1)
Experimental Basics	1112	(5)
Instrumentation	1112	(1)
Sample Preparation	1113	(1)
Collecting Data	1114	(1)
Anatomy of a Force Trace	1115	(1)
Detecting Intermediates in a Force Trace	1115	(1)
Analyzing the Force Trace	1116	(1)
Analysis of Force Data	1117	(12)
Basic Theory behind Dynamic Force Spectroscopy	1117	(2)
The Ramp of Force Experiment	1119	(2)
The Golden Equation of DFS	1121	(1)
Nonlinear Loading	1122	(1)
The Worm-line Chain (WLC)	1123	(1)
Experiments under Constant Force	1124	(1)
Effect of Tandem Repeats on Kinetics	1125	(1)
Determining the Modal Force	1126	(1)
Comparing Behavior	1127	(1)
Fitting the Data	1127	(2)
Use of Complementary Techniques	1129	(5)
Protein Engineering	1130	(1)
Choosing Mutants	1130	(1)
Determining ΔΔGD--N	1131	(1)
Determining ΔΔGTS--N	1131	(1)
Interpreting the Φ-values	1132	(1)
Computer Simulation	1133	(1)
Titin 127: A Case Study	1134	(5)
The Protein System	1134	(1)
The Unfolding Intermediate	1135	(1)
The Transition State	1136	(1)
The Relationship Between the Native and Transition States	1137	(2)
The Energy Landscape under Force	1139	(1)
Conclusions -- the Future	1139	(4)
References	1139	(4)
Molecular Dynamics Simulations to Study Protein Folding and Unfolding	1143	(27)
Amedeo Caflisch
Emanuele Paci
Introduction	1143	(1)
Molecular Dynamics Simulations of Peptides and Proteins	1144	(11)
Folding of Structured Peptides	1144	(1)
Reversible Folding and Free Energy Surfaces	1144	(3)
Non-Arrhenius Temperature Dependence of the Folding Rate	1147	(1)
Denatured State and Levinthal Paradox	1148	(1)
Folding Events of Trp-cage	1149	(1)
Unfolding Simulations of Proteins	1150	(1)
High-temperature Simulations	1150	(1)
Biased Unfolding	1150	(1)
Forced Unfolding	1151	(2)
Determination of the Transition State Ensemble	1153	(2)
MD Techniques and Protocols	1155	(7)
Techniques to Improve Sampling	1155	(1)
Replica Exchange Molecular Dynamics	1155	(2)
Methods Based on Path Sampling	1157	(1)
MD with Restraints	1157	(1)
Distributed Computing Approach	1158	(2)
Implicit Solvent Models versus Explicit Water	1160	(2)
Conclusion	1162	(8)
References	1162	(8)
Molecular Dynamics Simulations of Proteins and Peptides: Problems, Achievements, and Perspectives	1170
Paul Tavan
Heiko Carstens
Gerald Mathias
Introduction	1170	(1)
Basic Physics of Protein Structure and Dynamics	1171	(6)
Protein Electrostatics	1172	(1)
Relaxation Times and Spatial Scales	1172	(1)
Solvent Environment	1173	(1)
Water	1174	(1)
Polarizability of the Peptide Groups and of Other Protein Components	1175	(2)
State of the Art	1177	(10)
Control of Thermodynamic Conditions	1177	(1)
Long-range Electrostatics	1177	(2)
Polarizability	1179	(1)
Higher Multipole Moments of the Molecular Components	1180	(1)
MM Models of Water	1181	(1)
Complexity of Protein--Solvent Systems and Consequences for MM-MD	1182	(1)
What about Successes of MD Methods?	1182	(2)
Accessible Time Scales and Accuracy Issues	1184	(1)
Continuum Solvent Models	1185	(2)
Are there Further Problems beyond Electrostatics and Structure Prediction?	1187	(1)
Conformational Dynamics of a Light-switchable Model Peptide	1187
Computational Methods	1188	(2)
Results and Discussion	1190	(4)
Summary	1194	(1)
Acknowledgments	1194	(1)
References	1194
Contributors of Part II	lviii
Paradigm Changes from ``Unboiling an Egg'' to ``Synthesizing a Rabbit''	3	(29)
Rainer Jaenicke
Protein Structure, Stability, and Self-organization	3	(3)
Autonomous and Assisted Folding and Association	6	(5)
Native, Intermediate, and Denatured States	11	(2)
Folding and Merging of Domains -- Association of Subunits	13	(6)
Limits of Reconstitution	19	(2)
In Vitro Denaturation-Renaturation vs. Folding in Vivo	21	(3)
Perspectives	24	(8)
Acknowledgements	26	(1)
References	26	(6)
Folding and Association of Multi-domain and Oligomeric Proteins	32	(41)
Hauke Lilie
Robert Seckler
Introduction	32	(1)
Folding of Multi-domain Proteins	33	(8)
Domain Architecture	33	(2)
γ-Crystallin as a Model for a Two-domain Protein	35	(4)
The Giant Protein Titin	39	(2)
Folding and Association of Oligomeric Proteins	41	(13)
Why Oligomers?	41	(1)
Inter-subunit Interfaces	42	(2)
Domain Swapping	44	(1)
Stability of Oligomeric Proteins	45	(2)
Methods Probing Folding/Association	47	(1)
Chemical Cross-linking	47	(1)
Analytical Gel Filtration Chromatography	47	(1)
Scattering Methods	48	(1)
Fluorescence Resonance Energy Transfer	48	(1)
Hybrid Formation	48	(1)
Kinetics of Folding and Association	49	(1)
General Considerations	49	(1)
Reconstitution Intermediates	50	(2)
Rates of Association	52	(1)
Homo- Versus Heterodimerization	52	(2)
Renaturation versus Aggregation	54	(1)
Case Studies on Protein Folding and Association	54	(8)
Antibody Fragments	54	(5)
Trimeric Tail Spike Protein of Bacteriophage P22	59	(3)
Experimental Protocols	62	(11)
References	65	(8)
Studying Protein Folding in Vivo	73	(32)
I. Marije Liscaljet
Bertrand Kleizen
Ineke Braakman
Introduction	73	(1)
General Features in Folding Proteins Amenable to in Vivo Study	73	(6)
Increasing Compactness	76	(1)
Decreasing Accessibility to Different Reagents	76	(1)
Changes in Conformation	77	(1)
Assistance During Folding	78	(1)
Location-specific Features in Protein Folding	79	(5)
Translocation and Signal Peptide Cleavage	79	(1)
Glycosylation	80	(1)
Disulfide Bond Formation in the ER	81	(1)
Degradation	82	(1)
Transport from ER to Golgi and Plasma Membrane	83	(1)
How to Manipulate Protein Folding	84	(4)
Pharmacological Intervention (Low-molecular-weight Reagents)	84	(1)
Reducing and Oxidizing Agents	84	(1)
Calcium Depletion	84	(1)
ATP Depletion	85	(1)
Cross-linking	85	(1)
Glycosylation Inhibitors	85	(1)
Genetic Modifications (High-molecular-weight Manipulations)	86	(1)
Substrate Protein Mutants	86	(1)
Changing the Concentration or Activity of Folding Enzymes and Chaperones	87	(1)
Experimental Protocols	88	(17)
Protein-labeling Protocols	88	(1)
Basic Protocol Pulse Chase: Adherent Cells	88	(3)
Pulse Chase in Suspension Cells	91	(2)
(Co)-immunoprecipitation and Accessory Protocols	93	(1)
Immunoprecipitation	93	(1)
Co-precipitation with Calnexin ([84]; adapted from Ou et al. [85])	94	(1)
Co-immunoprecipitation with Other Chaperones	95	(1)
Protease Resistance	95	(1)
Endo H Resistance	96	(1)
Cell Surface Expression Tested by Protease	96	(1)
SDS-PAGE [13]	97	(1)
Acknowledgements	98	(1)
References	98	(7)
Characterization of ATPase Cycles of Molecular Chaperones by Fluorescence and Transient Kinetic Methods	105	(57)
Sandra Schlee
Jochen Reinstein
Introduction	105	(4)
Characterization of ATPase Cycles of Energy-transducing Systems	105	(1)
The Use of Fluorescent Nucleotide Analogues	106	(1)
Fluorescent Modifications of Nucleotides	106	(2)
How to Find a Suitable Analogue for a Specific Protein	108	(1)
Characterization of ATPase Cycles of Molecular Chaperones	109	(7)
Biased View	109	(1)
The ATPase Cycle of DnaK	109	(1)
The ATPase Cycle of the Chaperone Hsp90	109	(2)
The ATPase Cycle of the Chaperone ClpB	111	(1)
ClpB, an Oligomeric ATPase With Two AAA Modules Per Protomer	111	(1)
Nucleotide-binding Properties of NBD1 and NBD2	111	(3)
Cooperativity of ATP Hydrolysis and Interdomain Communication	114	(2)
Experimental Protocols	116	(46)
Synthesis of Fluorescent Nucleotide Analogues	116	(1)
Synthesis and Characterization of (Pβ)MABA-ADP and (Pγ)MABA-ATP	116	(3)
Synthesis and Characterization of N8-MABA Nucleotides	119	(1)
Synthesis of MANT Nucleotides	120	(1)
Preparation of Nucleotides and Proteins	121	(1)
Assessment of Quality of Nucleotide Stock Solution	121	(1)
Determination of the Nucleotide Content of Proteins	122	(1)
Nucleotide Depletion Methods	123	(1)
Steady-state ATPase Assays	124	(1)
Coupled Enzymatic Assay	124	(1)
Assays Based on [α-32P]-ATP and TLC	125	(1)
Assays Based on Released Pi	125	(1)
Single-turnover ATPase Assays	126	(1)
Manual Mixing Procedures	126	(1)
Quenched Flow	127	(1)
Nucleotide-binding Measurements	127	(1)
Isothermal Titration Calorimetry	127	(2)
Equilibrium Dialysis	129	(1)
Filter Binding	129	(1)
Equilibrium Fluorescence Titration	130	(2)
Competition Experiments	132	(1)
Analytical Solutions of Equilibrium Systems	133	(1)
Quadratic Equation	133	(1)
Cubic Equation	134	(4)
Iterative Solutions	138	(3)
Time-resolved Binding Measurements	141	(1)
Introduction	141	(1)
One-step Irreversible Process	142	(1)
One-step Reversible Process	143	(1)
Reversible Second Order Reduced to Pseudo-first Order	144	(2)
Two Simultaneous Irreversible Pathways -- Partitioning	146	(2)
Two-step Consecutive (Sequential) Reaction	148	(2)
Two-step Binding Reactions	150	(2)
References	152	(10)
Analysis of Chaperone Function in Vitro	162	(35)
Johannes Buchner
Stefan Walter
Introduction	162	(2)
Basic Functional Principles of Molecular Chaperones	164	(6)
Recognition of Nonnative Proteins	166	(1)
Induction of Conformational Changes in the Substrate	167	(2)
Energy Consumption and Regulation of Chaperone Function	169	(1)
Limits and Extensions of the Chaperone Concept	170	(2)
Co-chaperones	171	(1)
Specific Chaperones	171	(1)
Working with Molecular Chaperones	172	(2)
Natural versus Artificial Substrate Proteins	172	(1)
Stability of Chaperones	172	(2)
Assays to Assess and Characterize Chaperone Function	174	(2)
Generating Nonnative Conformations of Proteins	174	(1)
Aggregation Assays	174	(1)
Detection of Complexes Between Chaperone and Substrate	175	(1)
Refolding of Denatured Substrates	175	(1)
ATPase Activity and Effect of Substrate and Cofactors	176	(1)
Experimental Protocols	176	(21)
General Considerations	176	(1)
Analysis of Chaperone Stability	176	(1)
Generation of Nonnative Proteins	177	(1)
Model Substrates for Chaperone Assays	177	(2)
Suppression of Aggregation	179	(4)
Complex Formation between Chaperones and Polypeptide Substrates	183	(1)
Identification of Chaperone-binding Sites	184	(2)
Chaperone-mediated Refolding of Test Proteins	186	(2)
ATPase Activity	188	(1)
Acknowledgments	188	(1)
References	189	(8)
Physical Methods for Studies of Fiber Formation and Structure	197	(57)
Thomas Scheibel
Louise Serpell
Introduction	197	(1)
Overview: Protein Fibers Formed in Vivo	198	(8)
Amyloid Fibers	198	(1)
Silks	199	(1)
Collagens	199	(1)
Actin, Myosin, and Tropomyosin Filaments	200	(2)
Intermediate Filaments/Nuclear Lamina	202	(1)
Fibrinogen/Fibrin	203	(1)
Microtubules	203	(1)
Elastic Fibers	204	(1)
Flagella and Pili	204	(1)
Filamentary Structures in Rod-like Viruses	205	(1)
Protein Fibers Used by Viruses and Bacteriophages to Bind to Their Hosts	206	(1)
Overview: Fiber Structures	206	(7)
Study of the Structure of β-sheet-containing Proteins	207	(1)
Amyloid	207	(1)
Paired Helical Filaments	207	(1)
β-Silks	207	(1)
β-Sheet-containing Viral Fibers	208	(1)
α-Helix-containing Protein Fibers	209	(1)
Collagen	209	(1)
Tropomyosin	210	(1)
Intermediate Filaments	210	(1)
Protein Polymers Consisting of a Mixture of Secondary Structure	211	(1)
Tubulin	211	(1)
Actin and Myosin Filaments	212	(1)
Methods to Study Fiber Assembly	213	(15)
Circular Dichroism Measurements for Monitoring Structural Changes Upon Fiber Assembly	213	(1)
Theory of CD	213	(1)
Experimental Guide to Measure CD Spectra and Structural Transition Kinetics	214	(1)
Intrinsic Fluorescence Measurements to Analyze Structural Changes	215	(1)
Theory of Protein Fluorescence	215	(1)
Experimental Guide to Measure Trp Fluorescence	216	(1)
Covalent Fluorescent Labeling to Determine Structural Changes of Proteins with Environmentally Sensitive Fluorophores	217	(1)
Theory on Environmental Sensitivity of Fluorophores	217	(1)
Experimental Guide to Labeling Proteins With Fluorophores	218	(1)
1-Anilino-8-Naphthalensulfonate (ANS) Binding to Investigate Fiber Assembly	219	(1)
Theory on Using ANS Fluorescence for Detecting Conformational Changes in Proteins	219	(1)
Experimental Guide to Using ANS for Monitoring Protein Fiber Assembly	220	(1)
Light Scattering to Monitor Particle Growth	220	(1)
Theory of Classical Light Scattering	221	(1)
Theory of Dynamic Light Scattering	221	(1)
Experimental Guide to Analyzing Fiber Assembly Using DLS	222	(1)
Field-flow Fractionation to Monitor Particle Growth	222	(1)
Theory of FFF	222	(1)
Experimental Guide to Using FFF for Monitoring Fiber Assembly	223	(1)
Fiber Growth-rate Analysis Using Surface Plasmon Resonance	223	(1)
Theory of SPR	223	(1)
Experimental Guide to Using SPR for Fiber-growth Analysis	224	(1)
Single-fiber Growth Imaging Using Atomic Force Microscopy	225	(1)
Theory of Atomic Force Microscopy	225	(1)
Experimental Guide for Using AFM to Investigate Fiber Growth	225	(1)
Dyes Specific for Detecting Amyloid Fibers	226	(1)
Theory on Congo Red and Thioflavin T Binding to Amyloid	226	(1)
Experimental Guide to Detecting Amyloid Fibers with CR and Thioflavin Binding	227	(1)
Methods to Study Fiber Morphology and Structure	228	(13)
Scanning Electron Microscopy for Examining the Low-resolution Morphology of a Fiber Specimen	228	(1)
Theory of SEM	228	(1)
Experimental Guide to Examining Fibers by SEM	229	(1)
Transmission Electron Microscopy for Examining Fiber Morphology and Structure	230	(1)
Theory of TEM	230	(1)
Experimental Guide to Examining Fiber Samples by TEM	231	(1)
Cryo-electron Microscopy for Examination of the Structure of Fibrous Proteins	232	(1)
Theory of Cryo-electron Microscopy	232	(1)
Experimental Guide to Preparing Proteins for Cryo-electron Microscopy	233	(1)
Structural Analysis from Electron Micrographs	233	(1)
Atomic Force Microscopy for Examining the Structure and Morphology of Fibrous Proteins	234	(1)
Experimental Guide for Using AFM to Monitor Fiber Morphology	234	(2)
Use of X-ray Diffraction for Examining the Structure of Fibrous Proteins	236	(1)
Theory of X-Ray Fiber Diffraction	236	(1)
Experimental Guide to X-Ray Fiber Diffraction	237	(2)
Fourier Transformed Infrared Spectroscopy	239	(1)
Theory of FTIR	239	(1)
Experimental Guide to Determining Protein Conformation by FTIR	240	(1)
Concluding Remarks	241	(13)
Acknowledgements	242	(1)
References	242	(12)
Protein Unfolding in the Cell	254	(21)
Prakash Koodathingal
Neil E. Jaffe
Andreas Matouschek
Introduction	254	(1)
Protein Translocation Across Membranes	254	(6)
Compartmentalization and Unfolding	254	(2)
Mitochondria Actively Unfold Precursor Proteins	256	(1)
The Protein Import Machinery of Mitochondria	257	(2)
Specificity of Unfolding	259	(1)
Protein Import into Other Cellular Compartments	259	(1)
Protein Unfolding and Degradation by ATP-dependent Proteases	260	(6)
Structural Considerations of Unfoldases Associated With Degradation	260	(1)
Unfolding Is Required for Degradation by ATP-dependent Proteases	261	(1)
The Role of ATP and Models of Protein Unfolding	262	(1)
Proteins Are Unfolded Sequentially and Processively	263	(1)
The Influence of Substrate Structure on the Degradation Process	264	(1)
Unfolding by Pulling	264	(1)
Specificity of Degradation	265	(1)
Conclusions	266	(1)
Experimental Protocols	266	(9)
Size of Import Channels in the Outer and Inner Membranes of Mitochondria	266	(1)
Structure of Precursor Proteins During Import into Mitochondria	266	(1)
Import of Barnase Mutants	267	(1)
Protein Degradation by ATP-dependent Proteases	267	(1)
Use of Multi-domain Substrates	268	(1)
Studies Using Circular Permutants	268	(1)
References	269	(6)
Natively Disordered Proteins	275	(83)
Gary W. Daughdrill
Gary J. Pielak
Vladimir N. Uversky
Marc S. Cortese
A. Keith Dunker
Introduction	275	(6)
The Protein Structure-Function Paradigm	275	(2)
Natively Disordered Proteins	277	(3)
A New Protein Structure-Function Paradigm	280	(1)
Methods Used to Characterize Natively Disordered Proteins	281	(34)
NMR Spectroscopy	281	(1)
Chemical Shifts Measure the Presence of Transient Secondary Structure	282	(2)
Pulsed Field Gradient Methods to Measure Translational Diffusion	284	(1)
NMR Relaxation and Protein Flexibility	284	(1)
Using the Model-free Analysis of Relaxation Data to Estimate Internal Mobility and Rotational Correlation Time	285	(1)
Using Reduced Spectral Density Mapping to Assess the Amplitude and Frequencies of Intramolecular Motion	286	(1)
Characterization of the Dynamic Structures of Natively Disordered Proteins Using NMR	287	(1)
X-ray Crystallography	288	(5)
Small Angle X-ray Diffraction and Hydrodynamic Measurements	293	(4)
Circular Dichroism Spectropolarimetry	297	(2)
Infrared and Raman Spectroscopy	299	(2)
Fluorescence Methods	301	(1)
Intrinsic Fluorescence of Proteins	301	(1)
Dynamic Quenching of Fluorescence	302	(1)
Fluorescence Polarization and Anisotropy	303	(1)
Fluorescence Resonance Energy Transfer	303	(2)
ANS Fluorescence	305	(3)
Conformational Stability	308	(1)
Effect of Temperature on Proteins with Extended Disorder	309	(1)
Effect of pH on Proteins with Extended Disorder	309	(1)
Mass Spectrometry-based High-resolution Hydrogen-Deuterium Exchange	309	(2)
Protease Sensitivity	311	(2)
Prediction from Sequence	313	(1)
Advantage of Multiple Methods	314	(1)
Do Natively Disordered Proteins Exist Inside Cells?	315	(7)
Evolution of Ordered and Disordered Proteins Is Fundamentally Different	315	(1)
The Evolution of Natively Disordered Proteins	315	(2)
Adaptive Evolution and Protein Flexibility	317	(1)
Phylogeny Reconstruction and Protein Structure	318	(2)
Direct Measurement by NMR	320	(2)
Functional Repertoire	322	(6)
Molecular Recognition	322	(1)
The Coupling of Folding and Binding	322	(1)
Structural Plasticity for the Purpose of Functional Plasticity	323	(1)
Systems Where Disorder Increases Upon Binding	323	(2)
Assembly/Disassembly	325	(1)
Highly Entropic Chains	325	(2)
Protein Modification	327	(1)
Importance of Disorder for Protein Folding	328	(3)
Experimental Protocols	331	(27)
NMR Spectroscopy	331	(1)
General Requirements	331	(1)
Measuring Transient Secondary Structure in Secondary Chemical Shifts	332	(1)
Measuring the Translational Diffusion Coefficient Using Pulsed Field Gradient Diffusion Experiments	332	(1)
Relaxation Experiments	332	(1)
Relaxation Data Analysis Using Reduced Spectral Density Mapping	333	(1)
In-cell NMR	334	(1)
X-ray Crystallography	334	(2)
Circular Dichroism Spectropolarimetry	336	(1)
Acknowledgements	337	(1)
References	337	(21)
The Catalysis of Disulfide Bond Formation in Prokaryotes	358	(19)
Jean-Francois Collet
James C. Bardwell
Introduction	358	(1)
Disulfide Bond Formation in the E. coli Periplasm	358	(7)
A Small Bond, a Big Effect	358	(1)
Disulfide Bond Formation Is a Catalyzed Process	359	(1)
DsbA, a Protein-folding Catalyst	359	(2)
How is DsbA Re-oxidized?	361	(1)
From Where Does the Oxidative Power of DsbB Originate?	361	(1)
How Are Disulfide Bonds Transferred From DsbB to DsbA?	362	(2)
How Can DsbB Generate Disulfide by Quinone Reduction?	364	(1)
Disulfide Bond Isomerization	365	(4)
The Protein Disulfide Isomerases DsbC and DsbG	365	(1)
Dimerization of DsbC and DsbG Is Important for Isomerase and Chaperone Activity	366	(1)
Dimerization Protects from DsbB Oxidation	367	(1)
Import of Electrons from the Cytoplasm: DsbD	367	(2)
Conclusions	369	(1)
Experimental Protocols	369	(8)
Oxidation-reduction of a Protein Sample	369	(1)
Determination of the Free Thiol Content of a Protein	370	(1)
Separation by HPLC	371	(1)
Tryptophan Fluorescence	372	(1)
Assay of Disulfide Oxidase Activity	372	(1)
References	373	(4)
Catalysis of Peptidyl-prolyl cis/trans Isomerization by Enzymes	377	(38)
Gunter Fischer
Introduction	377	(2)
Peptidyl-prolyl cis/trans Isomerization	379	(4)
Monitoring Peptidyl-prolyl cis/trans Isomerase Activity	383	(5)
Prototypical Peptidyl-prolyl cis/trans Isomerases	388	(11)
General Considerations	388	(2)
Prototypic Cyclophilins	390	(4)
Prototypic FK506-binding Proteins	394	(3)
Prototypic Parvulins	397	(2)
Concluding Remarks	399	(1)
Experimental Protocols	399	(16)
PPIase Assays: Materials	399	(1)
PPIase Assays: Equipment	400	(1)
Assaying Procedure: Protease-coupled Spectrophotometric Assay	400	(1)
Assaying Procedure: Protease-free Spectrophotometric Assay	401	(1)
References	401	(14)
Secondary Amide Peptide Bond cis/trans Isomerization in Polypeptide Backbone Restructuring: Implications for Catalysis	415	(14)
Cordelia Schiene-Fischer
Christian Lucke
Introduction	415	(1)
Monitoring Secondary Amide Peptide Bond cis/trans Isomerization	416	(2)
Kinetics and Thermodynamics of Secondary Amide Peptide Bond cis/trans Isomerization	418	(2)
Principles of DnaK Catalysis	420	(3)
Concluding Remarks	423	(1)
Experimental Protocols	424	(5)
Stopped-flow Measurements of Peptide Bond cis/trans Isomerization	424	(1)
Two-dimensional 1H-NMR Exchange Experiments	425	(1)
References	426	(3)
Ribosome-associated Proteins Acting on Newly Synthesized Polypeptide Chains	429	(30)
Sabine Rospert
Matthias Gautschi
Magdalena Rakwalska
Uta Raue
Introduction	429	(3)
Signal Recognition Particle, Nascent Polypeptide--associated Complex, and Trigger Factor	432	(4)
Signal Recognition Particle	432	(3)
An Interplay between Eukaryotic SRP and Nascent Polypeptide--associated Complex?	435	(1)
Interplay between Bacterial SRP and Trigger Factor?	435	(1)
Functional Redundancy: TF and the Bacterial Hsp70 Homologue DnaK	436	(1)
Chaperones Bound to the Eukaryotic Ribosome: Hsp70 and Hsp40 Systems	436	(7)
Sis1p and Ssa1p: an Hsp70/Hsp40 System Involved in Translation Initiation?	437	(1)
Ssb1/2p, an Hsp70 Homologue Distributed Between Ribosomes and Cytosol	438	(1)
Function of Ssb1/2p in Degradation and Protein Folding	439	(1)
Zuotin and Ssz1p: a Stable Chaperone Complex Bound to the Yeast Ribosome	440	(1)
A Functional Chaperone Triad Consisting of Ssb1/2p, Ssz1p, and Zuotin	440	(2)
Effects of Ribosome-bound Chaperones on the Yeast Prion [PSI+]	442	(1)
Enzymes Acting on Nascent Polypeptide Chains	443	(2)
Methionine Aminopeptidases	443	(1)
Nα-acetyltransferases	444	(1)
A Complex Arrangement at the Yeast Ribosomal Tunnel Exit	445	(1)
Experimental Protocols	446	(13)
Purification of Ribosome-associated Protein Complexes from Yeast	446	(1)
Growth of Yeast and Preparation of Ribosome-associated Proteins by High-salt Treatment of Ribosomes	447	(1)
Purification of NAC and RAC	448	(1)
References	449	(10)
The Role of Trigger Factor in Folding of Newly Synthesized Proteins	459	(31)
Elke Deuerling
Thomas Rauch
Holger Patzelt
Bernd Bukau
Introduction	459	(1)
In Vivo Function of Trigger Factor	459	(6)
Discovery	459	(1)
Trigger Factor Cooperates With the DnaK Chaperone in the Folding of Newly Synthesized Cytosolic Proteins	460	(1)
In Vivo Substrates of Trigger Factor and DnaK	461	(2)
Substrate Specificity of Trigger Factor	463	(2)
Structure--Function Analysis of Trigger Factor	465	(6)
Domain Structure and Conservation	465	(3)
Quaternary Structure	468	(1)
PPIase and Chaperone Activity of Trigger Factor	469	(1)
Importance of Ribosome Association	470	(1)
Models of the Trigger Factor Mechanism	471	(2)
Experimental Protocols	473	(17)
Trigger Factor Purification	473	(2)
GAPDH Trigger Factor Activity Assay	475	(1)
Modular Cell-free E. coli Transcription/Translation System	475	(8)
Isolation of Ribosomes and Add-back Experiments	483	(2)
Cross-linking Techniques	485	(1)
References	485	(5)
Cellular Functions of Hsp70 Chaperones	490	(26)
Elizabeth A. Craig
Peggy Huang
Introduction	490	(2)
``Soluble'' Hsp70s/ J-proteins Function in General Protein Folding	492	(3)
The Soluble Hsp70 of E. coli, DanK	492	(1)
Soluble Hsp70s of Major Eukaryotic Cellular Compartments	493	(1)
Eukaryotic Cytosol	493	(1)
Matrix of Mitochondria	494	(1)
Lumen of the Endoplasmic Reticulum	494	(1)
``Tethered'' Hsp70s/J-proteins: Roles in Protein Folding on the Ribosome and in Protein Translocation	495	(3)
Membrane-tethered Hsp70/J-protein	495	(1)
Ribosome-associated Hsp70/J-proteins	496	(2)
Modulating of Protein Conformation by Hsp70s/J-proteins	498	(6)
Assembly of Fe/S Centers	499	(1)
Uncoating of Clathrin-coated Vesicles	500	(1)
Regulation of the Heat Shock Response	501	(1)
Regulation of Activity of DNA Replication-initiator Proteins	502	(2)
Cases of a Single Hsp70 Functioning With Multiple J-Proteins	504	(1)
Hsp70s/J-proteins -- When an Hsp70 Maybe Isn't Really a Chaperone	504	(3)
The Ribosome-associated ``Hsp70'' Sszl	505	(1)
Mitochondrial Hsp70 as the Regulatory Subunit of an Endonuclease	506	(1)
Emerging Concepts and Unanswered Questions	507	(9)
References	507	(9)
Regulation of Hsp70 Chaperones by Co-chaperones	516	(47)
Matthias P. Mayer
Bernd Bukau
Introduction	516	(1)
Hsp70 Proteins	517	(9)
Structure and Conservation	517	(2)
ATPase Cycle	519	(2)
Structural Investigations	521	(1)
Interactions With Substrates	522	(4)
J-domain Protein Family	526	(8)
Structure and Conservation	526	(4)
Interaction With Hsp70s	530	(2)
Interactions with Substrates	532	(2)
Nucleotide Exchange Factors	534	(6)
GrpE: Structure and Interaction with DnaK	534	(1)
Nucleotide Exchange Reaction	535	(1)
Bag Family: Structure and Interaction With Hsp70	536	(2)
Relevance of Regulated Nucleotide Exchange for Hsp70s	538	(2)
TPR Motifs Containing Co-chaperones of Hsp70	540	(4)
Hip	541	(1)
Hop	542	(1)
Chip	543	(1)
Concluding Remarks	544	(1)
Experimental Protocols	544	(19)
Hsp70s	544	(1)
J-Domain Proteins	545	(1)
GrpE	546	(1)
Bag-1	547	(1)
Hip	548	(1)
Hop	549	(1)
Chip	549	(1)
References	550	(13)
Protein Folding in the Endoplasmic Reticulum Via the Hsp70 Family	563	(54)
Ying Shen
Kyung Tae Chung
Linda M. Hendershot
Introduction	563	(1)
Bip Interactions with Unfolded Proteins	564	(3)
ER-localized DnaJ Homologues	567	(4)
ER-localized Nucleotide-exchange/releasing Factors	571	(1)
Organization and Relative Levels of Chaperones in the ER	572	(1)
Regulation of ER Chaperone Levels	573	(2)
Disposal of BiP-associated Proteins That Fail to Fold or Assemble	575	(1)
Other Roles of BiP in the ER	576	(1)
Concluding Comments	576	(1)
Experimental Protocols	577	(40)
Production of Recombinant ER Proteins	577	(1)
General Concerns	577	(1)
Bacterial Expression	578	(2)
Yeast Expression	580	(1)
Baculovirus	581	(2)
Mammalian Cells	583	(3)